This invention relates to novel 5-substituted picolinic acid compounds, and particularly to novel 5-substituted picolinic acid compounds produced by fermentation of a fungus Marasmiellus sp., which has been deposited as FERM BP-5735. This invention also relates to processes for producing the 5-substituted picolinic acid compounds, and a pharmaceutical composition comprising the same, which is usefull in the treatment of IL-1 and TNF mediated diseases.
Interleukin-l (IL-1) and tumor necrosis factor (TNF) are biological substances produced by a variety of cells, such as monocytes or macrophages. IL-1 and TNF have been demonstrated to mediate a variety of biological activities thought to be important in immunoregulation and other physiological conditions such as inflammation.
There are many disease states in which excessive or unregulated IL-1 production is implicated in exacerbating and/or causing the disease. These include rheumatoid arthritis, osteoartbritis, endotoxemia and/or toxic shock syndrome, other acute or chronic inflammatory disease states such as the inflammatory reaction induced by endotoxin or inflammatory bowel disease; tuberculosis, atherosclerosis, muscle degeneration, cachexia, psoriatic arthritis, Reiter""s syndrome, rheumatoid arthritis, gout, traumatic arthritis, mbella arthritis, and acute synovitis. Recent evidence also links IL-1 activity to diabetes (T. Mandrup-Poulsen et al., Allergy, 1985, 40, 424). The only IL-1 blocker available today is the natural IL-1 receptor antagonist (IL-1RA), which is easily metabolized in the bloodstream with a very short halfAife (E. V. Granowitz et al., Cytokine, 1992, 4, 353). Thus, active research has been carried out to develop stable, long-acting agents which can be taken by oral administration or by parenteral injections rather than by intravenous infusion, which is required for IL-1RA. A number of compounds as IL-1 receptor antagonists, IL-1 biosynthesis inhibitors, and IL-1 converting enzyme inhibitors have been known (C. C. George et al., Exp. Opin. Ther. Paten, 1996, 6 (1), 41).
Excessive or unregulated TNF production has also been implicated in mediating or exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusion injury, acquired immunodeficiency syndrome (AIDS), AIDS related complex (ARC), keloid formation, scar tissue formation, Crohn""s disease, ulcerative colitis, or pyresis. Although significant progress in developing potent TNF modulators has been achieved through the use of recombinantly derived proteins including monoclonal antibodies and soluble receptors, the development of biosynthesis inhibitors and receptor antagonists has been less successful. Recently a number of small molecule TNF modulators have been claimed. Most of them which specifically inhibit TNF production do so by increasing intracellular cyclic adenosine monophosphate (cAMP) which ultimately blocks TNF gene expression (Y. KATAKAMI et al., Immunology, 1988, 64, 719). The most important of these compounds are the rolipram and pentoxifylline-related phosphodiesterase IV (PDE IV) inhibitors which are being pursued by a number of pharmaceutical companies (A. BADGER et al., Circul. Shock, 1994, 44, 188). The ability of thalidomide to block TNF production contributes to its therapeutic properties in humans (E. P. SAMPAIO et al., J. Exp. Med, 1991, 73, 699). Recent studies suggest that cell-associated may be necessary for normal host defense mechanisms. This finding has added to the excitement concerning the identification of a unique metalloproteinase enyyme which is responsible for the proteolytic processing of TNF. Inhibitors of matrix metalloproteinase-related enzyme have appeared (K. M. MOHLER et al., Nature, 1994, 370,218).
Inhibitors of interleukin 1, 6 and 8 and TNF are described in PCT application US94/07969 which was published on Jan. 26 1995. The inhibitors of TNF are also described in PCT application US94/04950 which was published on Nov. 24 1994. Substituted picolinic acid compounds have been known to be produced by fungus. These include phenopicolinic acid (5-(4-hydroxylbenzyl)picolinic acid) (T. Nakamura et al., J. Antibiotics, 27:477-, 1975), fusaric acid (5-butylpicolinic acid)( H. Hidaka et al., J.Antibiotics, 22:228-, 1969), and fusarinolic acid (K. Steiner et al., Helv. Chim. Acta, 54:845-, 1971).
The object of the present invention is to provide novel 5-substituted picolinic acid compounds having an excellent activities for TNF and/or IL-1 biosynthesis inhibition and a pharmaceutically composition comprising the same. Another object is to provide processes for producing the novel 5-substituted picolinic acid compounds.
Accordingly, the present invention provides novel 5-substituted picolinic acid compounds of formula (I): 
or a pharmaceutically acceptable salt thereof,
wherein R1 and R2 are independently H, C2-C6 acyl or halo-substituted benzoyl; and R3 is xe2x80x94C(O)Oxe2x80x94C1-C6 alkyl, C(O)OH, CN, CONH2, CONHCH3,xc2x0 CON(CH3)2, 1-methyltetrazole or 2-methyltetrazole, with the proviso that when R1 is acetyl and R3 is methoxycarbonyl, R1 is not H; and that when R3 is CN, CONH2, CONHCH3, CON(CH3)2, 1-methyltetrazole or 2-methyltetrazole, R1 and R2 are H.
Preferred compounds of this invention are those of formula (I) shown above, wherein R3 is xe2x80x94C(O)Oxe2x80x94C1-C6 alkyl or C(O)OH, with the proviso that when R2 is acetyl and R3 is methoxycarbonyl, R1 is not H.
The present invention also provides a culture of Marasmiellus sp. FERM BP-5735, which is capable of producing the 5-substituted picolinic acid compounds, especially those of formula (I) wherein wherein R1 and R2 are H, and R3 is methoxycarbonyl (methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate), or R1 is acetyl, R2 is H, and R3 is methoxycarbonyl (methyl 5-(1-acetoxy-2-hydroxypropyl)2-pyridinecarboxylate).
Further, the present invention provides a process for producing the 5-substituted picolinic acid compounds of formula (X), which comprises cultivating a microorganism having identifying characteristics of Morasmiellus sp., FERM BP-5735, or a mutant or recombinant form thereof.
The present invention further provides a process comprising additional steps of isolating the 5-substituted picolinic acid compounds from the fermentation broth and chemically modifying the isolated compounds.
Also, the present invention provides a pharmaceutical composition for use in the treatment of IL-1 and/or TNF mediated diseases, which comprises the 5-substituted picolinic acid compounds of formula (1) wherein R1 and R2 are H; and R3 is methoxycarbonyl (methyl 5-(1,2-dihydroxypropyl)-2-pyridinecrbo xylate); R1 is acetyl; R2 is H; and R3 is methoxycarbonyl (methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate); R1 and R2 are H; and R3 is C(O)OH (5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid); or R1 and R2 are acetyl; and R3 is methoxycarbonyl (methyl 5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate); or pharmaceutically acceptable salt thereof in an amount effective in such treatments, and a pharmaceutically acceptable carrier.
Also, the present invention provides a method for the.treatment of IL-1 and/or TNF mediated diseases, which comprises administering to said subject an antinflammation amount of the compound of formula (1) wherein R1 and R2 are H; and R3 is methoxycarbonyl (methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate); R1 is acetyl; R2 is H; and R3 is methoxycarbonyl (methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate); R1 and R2 are H; and R3 is C(O)OH (5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid); or R1 and R2 are acetyl; and R3 is methoxycarbonyl (methyl 5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate); and, a pharmaceutically acceptable carrier.
The microorganism used in this invention is a strain of Marasmiellus sp. which was identified by and obtained from University of Tennessee. It was deposited under the accession number FERM BP-5735 to National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (located at 1-3 Higashi 1-chome, Tsukuba, Ibaraki 305, Japan) under the Budapest Treaty on Oct. 29, 1996. The taxonomical properties of the genus Marasmiellus have been reported by Singer, R. (in The Agaricales in modern taxonomy, 320-328, 1986).
In this invention, a mutant or recombinant form of FERM BP-5735 having the ability to produce the novel 5-substituted picolimic acid compounds of formula (I) can be also used. The mutant or recombinant form may be obtained by spontaneous mutation, artificial mutation with ultraviolet radiation, or treatment with mutagen such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine or ethyl methanesulfonate, or a cell technology method such as protoplast fusion, gene manipulation or the like, according to well-known methods.
According to the present invention, the novel 5-substituted picolinic acid compounds of formula (I) may be produced by aerobic fermentation of FERM BP-5735, or a mutant or recombinant form thereof, under conditions similar to those generally employed to produce bioactive compounds by fermentation.
FERM BP-5735, or a mutant or recombinant form thereof, is usually fermented under submerged aerobic conditions with agitation at a temperature of 20 to 40xc2x0 C. for 1 to 20 days, which may be varied according to fermentation conditions. Cultivation of FERM BP-5735 to produce said 5-substituted picolinic acid compounds of formula (1) preferably takes place in aqueous nutrient media at a temperature of 25 to 35xc2x0 C. for 10 to 15 days. The pH of medium may be adjusted in the range from 4.0 to 9.0, preferably from 5.5 to 7.0.
Nutrient media useful for fermentation include a source of assimilable carbon such as sugars, starches and glycerol; a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cotton seed meal, peanut meal, wheat gluten, soy flour, meat extract and fish meal; a source of growth substances such as mineral salts, sodium chloride and calcium carbonate; and trace elements such as iron, magnesium, copper, zinc, cobalt and manganese. If excessive foaming is encountered during fermentation, antifoam agents such as polypropylene glycols or silicones may be added to the fermentation medium.
Aeration of the medium in fermentors for submerged growth is maintained at 10 to 200%, preferably at 50 to 150% volumes of sterile air per volume of the medium per minutes. The rate of agitation depends on the type of agitator employed. A shake flask is usually run at 150 to 250 rpm whereas a fermentor is usually run at 300 to 2,000 rpm. Aseptic conditions must, of course, be maintained through the taansfer of the organism and throughout its growth.
The 5-substituted picolinic acid compounds thus produced may be isolated by standard techniques such as extraction and various chromatographic techniques.
As 5-substituted picolinic acid compounds of this invention, a compound of formula (I) wherein R1 and R2 are H, and R3 is methoxycarbonyl (methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate); and a compound of formula (1) wherein R1 is acetyl, R2 is H, and R3 is methoxycarbonyl (methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate) were isolated in a substantially pure form from the fermentation mixture. As 5-substituted picolinic acid compounds of this invention, there were synthesized 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid, methyl 5-(1,2diacetoxypropyl)2-pyridinecarboxylate, methyl 5-(1,2-i-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate, 5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide, 5-(1,2diydroxypropyl)-N,N-diethyl-2-pyridinecarboxamide, 5-(1,2dihydroxypropyl)-2-pyridinecarbonitrile, 5-(1,2-di-hydroxypropyl)2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine and 5-(1,2-di-hydroxypropyl)2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine from methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate by chemical modification. These compounds were identified by various spectroscopic techniques such as UV spectrophotometty, NMR andnass spectrometries, and the results will be shown in the section for working examples.
The compounds of formula (I) wherein R1 and R2 are an acyl group can be prepared by acylation of methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate, and the compounds of formula (I) wherein R3 is alkoxycarbonyl can be prepared by alkylation of demethyl methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate using suitable acylation and alkylating agents under suitable conditions known to those skilled in the art.
Preferred compounds of this invention include those of formula (I), wherein
R1 and R2 are H; and R3 is methoxycarbonyl (methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate);
R1 is acetyl; R2 is H; and R3 is methoxycarbonyl (methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate);
R1 and R2 are H; and R3 is C(O)OH (5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid);
R1 and R2 are acetyl; and R3 is methoxycarbonyl (methyl 5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate); and
R1 and R2 are p-bromobenzoyl; and R3 is methoxycarbonyl (methyl 5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate).
Preferred compounds of this invention also include those of the formula (I), wherein
R1 and R2 are H, and R3 is CN(5-(1,2dihydroxypropyl)-2-pyridinecarbonitrile);
R1 and R2 are H, and R3 is CONH2 (5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide);
R1 and R2 are H, and R3 is CONHCH3 (5-(1,2 dihydroxypropyl)-N-methyl-2-pyridinecarboxamide);
R1 and R2 are H, and R3 is CON(CH3)2 (5-(1,2dihydroxypropyl)-N,Nimethyl-2-pynidinecarboxamide);
R1 and R2 are H, and R3 is 1-methyltetrazole (5-(1,2-di-hydroxypropyl)-2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine); and ;
R1 and R2 are H, and R3 is 2-methyltetrazole (5-(1,2-di-hydroxypropyl)-2-(2-methyl-1,2,3,4-tetrazol-5-yl)pyridine).
More preferred compounds include a compound of formula (I), wherein
R1 and R2 are H; and R3 is methoxycarbonyl (methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate);
R1 is acetyl; R2 is H; and R3 is methoxycarbonyl (methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate);
R1 and R2 are H; and R3 is C(O)OH (5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid); and
R1 and R2 are acetyl; and R3 is methoxycarbonyl (methyl 5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate).
Further preferred -compounds of this invention also include those of formula (I) wherein
R1 and R2 are H, R3 is CONH2 (5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide)
R1 and R2 are H, R3 is CON(CH3)2 (5-(1,2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide)
R1 and R2 are H, R3 is CN (5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile)
R1 and R2 are H, R3 is 1-methyltetrazole (5-(1, 2-di-hydroxypropyl)-2-(2-methyl-2H-1,2,3,4tetrazol-5-yl)pyridine) and
R1 and R2 are H, R3 is 2-methyltetrazole (5-(1, 2-di-hydroxypropyl)-2-(1-methyl-1H-1,2,3 ,4-tetrazol-5-yl)pyridine).
The IL-1 and TNF biosynthesis inhibitory activities of the above-mentioned 5-substituted picolinic acid compounds, methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate, (methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate, 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid , methyl 5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate, methyl 5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate, 5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide, 5-(1, 2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide, 5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile, 5-(1,2-di-hydroxypropyl)-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine and 5-(1,2-di-hydroxypropyl)-2-(1-methyl-1H-1,2,3,4tetrazol-5-yl)pyridine), were measure by the standard in vitro protocol described below. These compounds were found to have the IL-1 and TNF biosynthesis inhibitory activities.
TNF Bioassay
Heparinised human whole blood diluted four-fold with RPMI medium was incubated with 10 xcexcg/ml of Lipopolysaccharide (LPS) in the presence of various concentrations of samples at 37xc2x0 C. in a humidified atmosphere containing 5% CO2 for 4 h. The TNF titer in the supernatants was determined with L929 cells which were destroyed by TNF quantitatively. L929 cells (2.5xc3x97104 cells) in 90 xcexcl of E-MEM medium containing 1% fetal calf serum and 0.5 xcexcg/ml of actinomycin D were placed in wells of 96-well microplates (flat-bottom). Ten xcexcl of the supernatants was added to each well and incubated at 37xc2x0 C. in a humidified atmosphere containing 5% CO2. After 18 h, the plates were rinsed with 0.9% sterile saline and stained for 10 min with 0.4% crystal violet in MeOH. The plates were washed with distilled water and were dried by air. Fifty xcexcl of methanol was added to each well to elute the crystal violet, and the plates were read on a microplate reader (model 3550, BIO-RAD) at 595 nm. TNF production inhibitory activity is calculated by the formula:       Inhibition    ⁢          xe2x80x83        ⁢          (      %      )        =            {              1        -                              [                                                            A                  595                                ⁢                                  xe2x80x83                                ⁢                Sample                            -                                                A                  595                                ⁢                                  xe2x80x83                                ⁢                Blank                                      ]                                [                                                            A                  595                                ⁢                                  xe2x80x83                                ⁢                Control                            -                                                A                  595                                ⁢                                  xe2x80x83                                ⁢                Blank                                      ]                              }        xc3x97    100  
IL-1 Bioassay
The supernatants prepared by the same method as TNF bioassay were analyzed IL-1 titer by commercially available specific ELISA system. The plates were read on a microplate reader (model 3550, BIO-RAD) at 490 nm. IL-1 production inhibitory activity is calculated by the formula:       Inhibition    ⁢          xe2x80x83        ⁢          (      %      )        =            {              1        -                              [                                                            A                  490                                ⁢                                  xe2x80x83                                ⁢                Sample                            -                                                A                  490                                ⁢                                  xe2x80x83                                ⁢                Blank                                      ]                                [                                                            A                  490                                ⁢                                  xe2x80x83                                ⁢                Control                            -                                                A                  490                                ⁢                                  xe2x80x83                                ⁢                Blank                                      ]                              }        xc3x97    100  
The pharmaceutically acceptable salts of methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate, methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate, 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid, methyl 5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate and methyl 5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate are prepared in a conventional manner by treating a solution or suspension of the compound with about one chemical equivalent of a pharmaceutically acceptable acid. Conventional concentration and recrystallization techniques are employed in isolating the salts.
Administration
The 5-substituted picolinic acid compounds of formula (I) a pharmaceutically acceptable salt are useful in the treatment of inflammation or the like. The 5-substituted picolinic acid compounds of formula (I) and a pharmaceutically acceptable salt may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. The pharmaceutical compositions formed by combining the 5-substituted picolinic acid compounds of formula (I) and the pharmaceutically acceptable carriers are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch, alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the essential active ingredients therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof.
For parenteral administration, solutions of the 5-substituted picolinic acid compounds of formula (I) and a pharmaceutically acceptable salt in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed. Such aqueous solutions should be suitable buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitioneal administration. In this connection, the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.
Additionally, the 5-substituted picolinic acid compounds of formula (I) and a pharmaceutically acceptable salt may be administered topically when treating conditions of the skin and this may be done by way of creams, jellies, gels, pastes, and ointments, in accordance with standard pharmaceutical practice.
In general, the 5-substituted picolinic acid compounds of formula (I) or its pharmaceutically acceptable salt are present in the above dosage forms at concentration levels ranging 5 to 70% by weight, preferably 10 to 50% by weight.
In general, a therapeutically effective daily dose for the active compound will range from 0.01 to 100 mg/kg, generally from about 1 to about 5 mg/kg As is generally known, the effective dosage for the active compound depends on the intended route of administration and other factors such as age and weight of the patient, as generally known to a physician. The dosage also depends on the illness to be treated.